Abstract
Ethylene is an important plant hormone that regulates numerous cellular processes and stress responses. The mode of action of ethylene is both dose- and time-dependent. Protein phosphorylation plays a key role in ethylene signaling, which is mediated by the activities of ethylene receptors, constitutive triple response 1 (CTR1) kinase, and phosphatase. To address how ethylene alters the cellular protein phosphorylation profile in a time-dependent manner, differential and quantitative phosphoproteomics based on (15)N stable isotope labeling in Arabidopsis was performed on both one-minute ethylene-treated Arabidopsis ethylene-overly-sensitive loss-of-function mutant rcn1-1, deficient in PP2A phosphatase activity, and a pair of long-term ethylene-treated wild-type and loss-of-function ethylene signaling ctr1-1 mutants, deficient in mitogen-activated kinase kinase kinase activity. In total, 1079 phosphopeptides were identified, among which 44 were novel. Several one-minute ethylene-regulated phosphoproteins were found from the rcn1-1. Bioinformatic analysis of the rcn1-1 phosphoproteome predicted nine phosphoproteins as the putative substrates for PP2A phosphatase. In addition, from CTR1 kinase-enhanced phosphosites, we also found putative CTR1 kinase substrates including plastid transcriptionally active protein and calcium-sensing receptor. These regulatory proteins are phosphorylated in the presence of ethylene. Analysis of ethylene-regulated phosphosites using the group-based prediction system with a protein-protein interaction filter revealed a total of 14 kinase-substrate relationships that may function in both CTR1 kinase- and PP2A phosphatase-mediated phosphor-relay pathways. Finally, several ethylene-regulated post-translational modification network models have been built using molecular systems biology tools. It is proposed that ethylene regulates the phosphorylation of arginine/serine-rich splicing factor 41, plasma membrane intrinsic protein 2A, light harvesting chlorophyll A/B binding protein 1.1, and flowering bHLH 3 proteins in a dual-and-opposing fashion.
Highlights
From the ‡Division of Life Science, The Hong Kong University of Science and Technology, Hong Kong SAR, China; ¶Department of Chemical and Biomolecular Engineering, The Hong Kong University of Science and Technology, Hong Kong SAR, China; ʈDepartment of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei 430074, China; **State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
A phosphatase loss-of-function mutant rcn1-1 was employed because both the sensitivity and the amplitude of the ethylene response are enhanced in this mutant (Fig. 1A), which may lead to easier detection of rapidly ethylene-induced phosphoproteins, whereas in the case of long-term ethylene-altered receptor complexes and cellular phosphor-relay networks, we focused on the identification of both direct and indirect putative in vivo substrates of constitutive triple response 1 (CTR1) kinase
15N Stable Isotope Labeling and Ethylene Treatment of Arabidopsis—The wild-type plant and two loss-of-function ethylene response mutants, ctr1-1 and rcn1-1, were selected for quantitative phosphoproteomics to study the phosphoproteomes of time-dependent ethylene responses in Arabidopsis
Summary
Plant Growth, 15N Stable Isotope Labeling, and Ethylene Treatment—The wild-type Arabidopsis thaliana ecotypes Columbia-0 (Col-0) and ctr mutant were obtained from the Arabidopsis Biological Resource Center (Columbus, OH); eer and rcn mutants were gifts from Dr Caren Chang and from Dr Alison DeLong and Dr Gloria K. A second set of IMAC beads made of TiO2 was employed In this IMAC protocol, 800 l of TiO2 loading buffer containing 1.25 M glycolic acid, 6% trifluoroacetic acid (TFA), and 82.5% ACN was mixed with the flow-through fractions from Fe3ϩ-NTA IMAC, and the flow-through peptides were incubated with 5 mg of TiO2 beads (GL Science Inc., Tokyo, Japan) for another 45 min. To further eliminate the random variation resulting from the protein mixing, the median value mr of the log ratios of ion intensities of 14N/15N-coded phosphopeptide pairs was generated based on each experimental data set (either forward or reciprocal). The resulting mixtures containing 0-, 1-, 5- and 15-min ethylene-treated kinase extracts were lyophilized and incubated with iTRAQ 4-plex labeling buffer (Applied Biosystems, Foster City, CA) that contained isobaric reagents providing a chemical linker of m/z 114, 115, 116, and 117 in MS acquisition, respectively, for 1 h at room temperature as previously described [25]. D, overall morphology of ctr plants, the loss-of-function line of CTR1 protein, a Raf-like kinase, and a member of the MAPKKK family that is thought to be a negative regulator in the ethylene signaling pathway, grown in the naturally 14N-abundant medium (left) or a medium enriched with
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